Spectroscopic and Crystallographic Evidence for the Role of a Water-Containing H-Bond Network in Oxidase Activity of an Engineered Myoglobin

Abstract

Heme-copper oxidases (HCOs) catalyze efficient reduction of oxygen to water in biological respiration. Despite progress in studying native enzymes and their models, the roles of non-covalent interactions in promoting this activity are still not well understood. Here we report EPR spectroscopic studies of cryoreduced oxy-F33Y-CuBMb, a functional model of HCOs engineered in myoglobin (Mb). We find that cryoreduction at 77 K of the O2-bound form, trapped in the conformation of the parent oxyferrous form, displays a ferric-hydroperoxo EPR signal, in contrast to the cryoreduced oxy-wild-type (WT) Mb, which is unable to deliver a proton and shows a signal from the peroxo-ferric state. Crystallography of oxy-F33Y-CuBMb reveals an extensive H-bond network involving H2O molecules, which is absent from oxy-WTMb. This H-bonding proton-delivery network is the key structural feature that transforms the reversible oxygen-binding protein, WTMb, into F33Y-CuBMb, an oxygen-activating enzyme that reduces O2 to H2O. These results provide direct evidence of the importance of H-bond networks involving H2O in conferring enzymatic activity to a designed protein. Incorporating such extended H-bond networks in designing other metalloenzymes may allow us to confer and fine-tune their enzymatic activities.

Activation of dioxygen by copper metalloproteins and insights from model complexes

“…Hydrogen bonding to, or protonation of, the peroxide may favor heterolytic, rather than homolytic, O-O bond cleavage. Recently, evidence for the H-bonding network in iron-only F33Y-Cu B Mb was shown using EPR and 1 H-ENDOR spectroscopies, and X-ray crystallography, illustrating the interaction of discrete water molecules with the oxy-F33Y-Cu B Mb [236]. Further modification of the active site through incorporation of unnatural amino acid imiTyr, a tyrosine derivative containing an imidazole in the ortho position on the phenol ring, yielded imiTyrCu B Mb, which contains an active site Tyr-His cross-link (Fig.…”

Critical Aspects of Heme–Peroxo–Cu Complex Structure and Nature of Proton Source Dictate Metal–Operoxo Breakage versus Reductive O–O Cleavage Chemistry

“…To provide the insights and fundamentals needed for this very important reaction, several different approaches can be taken. Studies on a diverse, ever-growing family of model complexes (both synthetic coordination complexes 11,36–38 and modified protein systems 12,22,23,39 ) have aided researchers in gaining a fundamental understanding about certain aspects of the chemical mechanism of O 2 reduction. 12,14 Our research group has for many years been systematically studying the O 2 adducts of synthetic model systems in which we can define or control certain aspects and monitor the effects of varying coordination environments/geometries, redox properties, sterics of the Cu and/or iron centers, and nature of a given substrate on the outcomes of different reactions.…”

Design of Heteronuclear Metalloenzymes

“…General purification of our designed heteronuclear metalloproteins will not be discussed in great detail here, but generally falls into two categories: inclusion body and cytosolic purification. The inclusion body protocol is used extensively in models such as Cu B Mb and Fe B Mb and has been covered previously in those works (Springer & Sligar, 1987, Sigman et al, 2000, Sigman et al, 2000, Sigman et al, 2003, Pfister et al, 2005, Zhao et al, 2005, Zhao et al, 2006, Yeung et al, 2009, Lin et al, 2010, Lin et al, 2010, Miner et al, 2012, Chakraborty et al, 2014, Bhagi-Damodaran et al, 2014, Petrik et al, 2016). Additionally, cytosolic purification of C c P and its rationally designed mutants has also been described in great detail by our group (Yeung et al, 1997, Sigman et al, 1999, Feng et al, 2003, Pfister et al, 2007, Miner et al, 2014, Hosseinzadeh et al, 2016).…”

“…The secondary interactions can confer their roles in many ways: engaging in a hydrogen bond or salt bridge to the primary ligands, changing the overall electrostatic environment of the metal binding site by changing charge or hydrophobicity, engaging in proton or electron transfer, or providing space for the metal cofactors (Hosseinzadeh & Lu, 2015). The stability and reactivity of heteronuclear metal-binding sites depends heavily on secondary sphere interactions that may contribute to electronic coupling between metal cofactors, the redox potential (E∘′) of one or both metals, and the stabilization of substrates and reactive intermediates (Yikilmaz et al, 2002, Jackson & Brunold, 2004, Marshall et al, 2009, Berry et al, 2010, Shook & Borovik, 2010, New et al, 2012, Petrik et al, 2016). Engineering such sites into easily expressible protein scaffolds makes the process of studying an isolated heteronuclear site as well as its secondary coordination sphere less complicated by excluding the complex global effects present in native enzymes while working on a stable, three-dimensional platform that can be altered rationally.…”